Pifithrin-α decreases the radioprotective efficacy of a Podophyllum hexandrum (Himalayan mayapple) fraction (REC-2006) in HepG2 cells

Biotechnol. Appl. Biochem. (2009) 54, 53–64 (Printed in Great Britain) doi:10.1042/BA20080250 53

Pifithrin-α decreases the radioprotective efficacy of aPodophyllum hexandrum (Himalayan mayapple) fraction(REC-2006) in HepG2 cells

Pankaj Kumar Singh*, Raj Kumar*, Ashok Sharma*, Rajesh Arora*,Swatantra Kumar Jain† and Rakesh Kumar Sharma*1

*Division of Radiation Biology and Radioprotection, Institute of Nuclear Medicine and Allied Sciences (INMAS), Brig. S.K.Mazumdar Road, Delhi-110 054, India, and †Department of Biotechnology, Jamia Hamdard, Hamdard Nagar,Delhi-110 062, India

Inhibition of the tumour suppressor p53 by PFT(pifithrin-α) promotes p53-mediated apoptosis andprotects against doxorubicin-induced apoptosis. Thepresent study was carried out to evaluate theeffect of PFT on the radioprotective potential ofPodophyllum hexandrum fraction (REC-2006) in HepG2(p53++) cell line. REC-2006 (10−5 μg/ml) treatmentat 2 h before irradiation (10 Gy) rendered 80 +− 3%protection in HepG2 cells, whereas PFT debilitatedthe radioprotective potential of REC-2006. REC-2006increased the expression of Hsp70 (heat-shockprotein 70), HSF1 (heat-shock factor 1) and Bcl-2in irradiated HepG2 cells, whereas PFT whentreated with REC-2006 decreased the expression ofHsp70, HSF1 and Bcl-2 in HepG2 cells. REC-2006facilitated post-irradiation DNA repair by pausingcell-cycle progression at G1- and G2-phase, whereasno such cell-cycle arrest was observed in irra-diated HepG2 cells pretreated with PFT in irr-adiated HepG2 cells. No change was observed in Mdm2(murine double minute 2) and Ras-GAP (Ras-GTPase-activating protein) expression with or without PFTtreatment. Decrease in the expression of caspase 3and Bax was observed in HepG2 cells when REC-2006 treatment was given 2 h before irradiation;however, PFT treatment increased the expressionof Bax leading to apoptosis. It can be concludedthat p53 expression plays a major role in theREC-2006-mediated protection against acute irradi-ation in HepG2 cells. PFT treatment reduced theradioprotective efficacy of REC-2006 by inhibitingthe expression of HSF1 and Hsp70 and thereby theexpression of Bcl-2, by up-regulating the cell-cycle-regulatory proteins and therefore reducing the span oftime for DNA repair and also by inducing Bax-mediatedapoptosis. PFT did not, however, show any effect onp53 regulating protein (Mdm2) and pro-survival protein(Ras-GAP).

Introduction

Ionizing radiation induces severe damage in various cellularcompartments, ultimately leading to cell death. Severalintracellular proteins work in an orchestrated manner toprotect cells from genotoxic stress. The stress proteins[Hsps (heat-shock proteins)] are encoded by genes whoseexpression is substantially increased under stress conditions.Consequently, Hsps assist in the recovery from stress,either by repairing damaged proteins (protein refolding) orby degrading the damaged proteins [1]. Hsp70 has beenshown to act as an inhibitor of apoptosis [2]. Progression ofcell cycle is promoted by cyclins and their respective CDKs(cyclin-dependent kinases); however, perturbation in cell-cycle checkpoint regulation induces genomic instability incells [3]. Activation of the tumour suppressor p53 inducesgrowth arrest at the G1/S-phase boundary by inducingthe expression of the Waf-1/Cip-1 gene or induces theoccurrence of apoptosis in damaged cells, for the preventionof further proliferation of genetically damaged cells [4].p21waf-1/cip-1, one of the kinase group of CDK inhibitors,negatively regulates the cell cycle progression in the G1–S-phase by reducing the activity of cyclin E–CDK2 and cyclinD–CDK4 complexes [4].

DNA damage is known to result in the accumulationand activation of p53 protein, which has a pivotal role indeciding the fate of the cells by co-ordinating cell-cycle

Key words: apoptosis, radioprotection, HepG2 cell, Podophyllum hexandrum(Himalayan mayapple), pifithrin-α, Ras-GTPase-activating protein.

Abbreviations used: AIF, apoptosis-inducing factor; BCIP, 5-bromo-4-chloroindol-3-yl phosphate; DMF, dose modification factor; NBT, NitroBlue Tetrazolium; CDK, cyclin-dependent kinase; FBS, fetal bovine serum;HSF1, heat-shock factor 1; Hsp70, heat-shock protein 70; Mdm2, murinedouble minute 2; MEM, minimal essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PFT, pifithrin-α;pRb, retinoblastoma protein; Ras-GAP, Ras-GTPase-activating protein; TBS,Tris-buffered saline.

1 To whom correspondence should be addressed ([email protected] or [email protected]).

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54 P. K. Singh and others

arrest, managing apoptotic events, and by modulating theDNA repair processes [5]. Although the role of p53 intumour suppression and apoptosis is well established [6], itsspecific contribution to DNA repair and cell survival has alsobeen reported [7,8]. The role of PFT (pifithrin-α) as a p53inhibitor is well established [9–11]. Previously, PFT has beenshown to promote the p53-mediated apoptosis in JB6 cells[10] and to protect against doxorubicin-induced apoptosis[11]. The present study was carried out to evaluate theeffect of PFT on radiation-induced apoptosis and radiationprotection mediated by a Podophyllum hexandrum (Himalayanmayapple; Podophyllaceae family) fraction (REC-2006).

P. hexandrum, a herb native to the Himalayanregion, has been extensively investigated previously for itsradioprotective efficacy [12–19]. The explored mechanismsby which P. hexandrum extract offers radioprotectioninclude antioxidant activity [12–14], free-radical-scavengingactivity [14,15], anti-(lipid peroxidation) activity [15] andby modulation of vital proteins associated with stress,apoptosis and DNA repair [16–18]. The P. hexandrumchloroform fraction (REC-2006) used in the present studyhas been reported to be rich in lignans, particularlypodophyllotoxin and its derivatives [14]. Even if severalmechanisms have been proposed by which P. hexandrumextract renders radioprotection, however, the effect of PFTon radioprotective efficacy of REC-2006 and the modulationof proteins expression associated with radioprotectionhas not, to our knowledge, been investigated so far.Therefore, the present study was carried out to explorethe significance of p53 expression/inhibition, cell-cycleregulation and expression of stress-responsive gene inradioprotection rendered by a P. hexandrum fraction (REC-2006) using wild-type functional p53-gene-carrying HepG2cell line as an experimental model.

Materials and methods

ReagentsAll reagents used were of analytical grade. The culture me-dium MEM (minimal essential medium), antibiotics(penicillin G and streptomycin), trypsin and FBS (fetalbovine serum) were from HiMedia and Sigma–Aldrich.Mouse monoclonal antibodies for the detection ofhuman p53, Mdm2, Hsp70, HSF1, Bcl-2, Ras-GAP (Ras-GTPase-activating protein), cyclin D1, CDK4, cyclinB1, p21, caspase 3, Bax and β-actin proteins, andalkaline phosphatase-conjugated secondary anti-mouseantibodies were from Santa Cruz Biotechnology (SantaCruz, CA, U.S.A.). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], DTT (dithiothreitol),EDTA, PMSF, Nonidet P40, BCIP (5-bromo-4-chloroindol-3-yl phosphate)/NBT (Nitro Blue Tetrazolium) reagent

and protease inhibitors were from Sigma–Aldrich(St Louis, MO, U.S.A.). Lysine, Tris base and SDS wereobtained from Merck (Darmstadt, Germany), whereasnitrocellulose membrane was purchased from Millipore.

Plant extractDried rhizomes of P. hexandrum Royale growing at an altitudeof 4000 m in the Himalayan region were provided by theDefence Institute of High Altitude Research at Leh, Jammuand Kashmir, India. Dried powder (10 gm/100 ml, w/v) ofP. hexandrum rhizome was transferred to soxhlet apparatusand extracted three times with the solvents (1:6 ratio)of increasing polarity, namely hexane, chloroform, ethanol,50% ethanol in water and water, subsequently for over thecourse of 24–72 h. The respective filtrates were combined[14]. All the extracts were filtered through Whatman filterpaper no. 3 followed by filtration through a 0.22-μm-pore-size filter (Millipore). Extracts were then concentrated bysolvent evaporation under reduced pressure in a rotaryevaporator (Buchi, Flawil, Switzerland) and dried. Out offive different extracts prepared the dried chloroform extract(REC-2006) was used for the present study.

Cell cultureThe human hepatoma cell lines HepG2 (p53++) werepurchased from the NCCS (National Centre for CellScience, Pune, India). Cells were maintained in 25 cm2

culture flasks as monolayer culture in MEM supplementedwith 10% (v/v) FBS, 100 units/ml of penicillin and 100 μg/mlof streptomycin (pH 7.4) at 37 ◦C in a humidified 5% (v/v)CO2 incubator (Binder, Tuttlingen, Germany) in 25 cm2

culture flasks (Nunc, Rochester, NY, U.S.A.) [20]. Cells weresubcultured twice a week.

Harvesting of adherent cellsCells were harvested using 0.25% (w/v) trypsin in HBSS(Hanks balanced salt solution). In brief, after removing themedium, 1.0 ml of chilled trypsin (0.25%, w/v) was pouredinto the culture flask (T25) and left for 30 s at roomtemperature (25+−1 ◦C). After decanting excess trypsin, theculture flasks were incubated at 37 ◦C until the cells starteddetaching from the culture flask. Harvested cells werecounted and proper dilutions were made by addingcomplete MEM medium to the cell suspension for furtherexperimentation.

IrradiationA 60Co gamma chamber (Model 220; Atomic Energy ofCanada) was used to deliver desired radiation doses (doserate 43.8 cGy/min). Cells were cultured in culture flasks(25 cm2) and irradiated at different doses (5–12 Gy). Culture

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Pifithrin-α-mediated decrease in radioprotection 55

flasks fitted with a filter cap (Nunc) were used to avoid thegeneration of hypoxic conditions in the irradiation chamber.

MTT assayCell proliferation was detected by the MTT assay. Whencells were cultured to exponential phase, they wereseeded on a 96-well plate (2 × 104 cells/100 μl per well)for 24 h. Cells were divided into a control (DMSO)group and an REC-2006 group. The concentration ofREC-2006 added was 101–10−7 μg/ml of final volume. After60 h of incubation, 10 μl of MTT (5 g/l) was added andincubated at 37 ◦C for 4 h. DMSO (75 μl) was added toeach well, and the plate was oscillated for 10 min untilthe crystals were dissolved completely. Absorbance (A)was detected with an enzyme calibrator at 560 nm. Cellviability was calculated by using the following equation:

Cell viability = A 560of study groupA 560of control group

× 100 %

The experiment was repeated three times [20].

Experimental planThe HepG2 cells were divided into the following four groupswith and without PFT in triplicate.

Group I: untreated control of HepG2 (p53++) cellsGroup II: hepg2 cells treated with different concentrations(10−3–10−7 μg/ml) of REC-2006Group III: hepg2 cells exposed to different radiation doses(5–12 Gy)Group IV: hepg2 cells exposed to radiation doses 2 h afterREC-2006 (10−3–10−7 μg/ml) treatment

After irradiation, culture medium (MEM) was replacedwith the fresh REC-2006-free complete MEM culturemedium and cells were further incubated at 37 ◦C with5% CO2 in a humidified condition for 15 days. Colonieswere counted after the incubation period and percentagesurvival was calculated. All experiments were carried out intriplicate.

Similarly, HepG2 cells were divided into the four above-mentioned treatment groups and, after final treatment, cellswere harvested at 2, 4, 8 and 10 h for the comet assay andat 8 h for protein expression studies.

Effect of PFT (p53 inhibitor) on radioprotectionoffered by REC-2006 in HepG2 cellsTo evaluate the effect of p53 inhibition on REC-2006-mediated radioprotection, PFT-pretreated HepG2 cellswere incubated with the p53 transcriptional inhibitor PFT(10 mM) for 12 h at 37 ◦C, 5% CO2 and 90% humidity andwere further treated with REC-2006 for 2 h. After treat-

ment, the culture medium was replaced with fresh mediumand subjected to irradiation (10 Gy). The cells were thenallowed to grow for 15 days and the colonies were countedafter incubation.

DNA fragmentation assayTo evaluate the effect of irradiation and REC-2006 treatmenton human HepG2 cells, a single-cell gel electrophoresis assaywas performed [21]. In brief, cells of different treatmentgroups were trypsinized, washed with PBS and mixed withpre-warmed 0.75% (w/v) (500–600 μl) ultra-low gellingagarose (Sigma–Aldrich) and layered on microscopic slidesprecoated with 0.1% normal agarose. Slides were incubatedat 4 ◦C to allow the formation of agarose gel. Slideswere then submerged in the lysis buffer (2.5% SDS, 1 %sodium sarcosinate and 25 mM EDTA, pH 9.5) for 15 minat 25 ◦C. Slides were washed in distilled water for 5 min at10 ◦C. Electrophoresis was carried out in electrophoresisbuffer (90 mM Trizma base, 90 mM boric acid and 2.5 mMEDTA, pH 8.3) at 2 V/cm for 5 min. After a brief rinse indistilled water, slides were dried at 45 ◦C and stored in acool humid box until use. After rehydration of slidesin distilled water for 5 min, comets were stained withpropidium iodide (25 μM in PBS) and observed under afluorescence microscope (Olympus Optical, Tokyo, Japan).Analysis of comet was performed using comet scoresoftware (TriTek Comet Score version 1.5.0).

Extraction of cytoplasmic and nuclear proteinfractionsHepG2 cells were harvested by trypsinization and centri-fuged at 100 g for 5 min. The pellet was resuspendedin 300 μl of buffer A (50 mM NaCl, 10 mM Hepes,pH 8.0, 500 mM sucrose, 1 mM EDTA, 0.5 mM spermidine,0.15 mM spermine and 0.2% Triton X-100) containing2-mercaptoethanol and the protease inhibitors PMSF,leupeptin, aprotinin and pepstatin. After 15 min of incubationon ice, the suspension was centrifuged at 1400 g for 10 min.The supernatant (cytoplasmic fraction) was collected andstored, while the pellet was washed with 200 μl of buffer B(50 mM NaCl, 10 mM Hepes, pH 8, 25% glycerol, 0.1 mMEDTA, 0.5 mM spermidine and 0.15 mM spermine) and thenresuspended in 100 μl of buffer C (350 mM NaCl, 10 mMHepes, 25% glycerol, 0.1 mM EDTA, 0.5 mM spermidineand 0.15 mM spermine). After centrifugation at 17000 g thesupernatant (nuclear fraction) was collected and stored at4 ◦C [22].

Protein estimationTotal soluble protein contents in the different nuclearand cytoplasmic fractions were estimated as describedpreviously [17]. Briefly, 10 μl of the sample was mixed

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56 P. K. Singh and others

with 90 μl of distilled water. After thorough mixing,Bradford reagent (1.0 ml) was added and the absorbance wasrecorded at room temperature within 20–30 min at 595 nmusing a microtitre plate reader (Wallac). The amount (μg)of protein was quantified using a BSA standard curve.

SDS/PAGE analysis of proteinsFor SDS/PAGE analysis of the different protein fractions,10% polyacrylamide gels of 0.75 mm thickness wereprepared [17]. Protein fractions were added to SDS/PAGEsample buffer [0.0625 M Tris/HCl, pH 6.8, 2% (w/v) SDS, 5%(v/v) glycerol, 2% (v/v) 3-mercaptoethanol and 0.01% (w/v)Bromophenol Blue] and heated in a boiling-water bath for2–3 min. Equal amounts of protein samples (10 μg) wereloaded in each well. Electrophoresis was carried out at aconstant voltage (stacking at 60 V, resolving at 70 V). Afterelectrophoresis, the gels were stained with gentle shakingin 0.1% Coomassie Brilliant Blue R-250 in methanol/aceticacid/water (4:2:4, by vol.) at room temperature anddestained in a washing solution [methanol/acetic acid/water(1:0.7:8.3)] to obtain distinct bands over a clear background.The gels were stored in 0.1% acetic acid for future analysis.

Western-blot analysisFor Western-blot analysis, proteins were transferred elec-trophoretically (100 V, 1 h) on to a nitrocellulose membraneusing a Mini-Trans-Blot assembly (Bio-Rad, Hercules, CA,U.S.A.). The nitrocellulose membrane was blocked ina blocking solution [containing 5%, w/v, non-fat driedskimmed milk powder in TBS (Tris-buffered saline; 25 mMTris, 150 mM NaCl and 2 mM KCl, pH 7.4)] for 2 h atroom temperature. The expression levels of p53, Mdm2,Hsp70, HSF1, Bcl-2, Ras-GAP, cyclin D1, CDK4, cyclin B1,p21, caspase 3, Bax and β-actin were analysed by probingwith respective mouse monoclonal antibodies (1:1000dilution) having cross-reactivity with human proteins. Afterthree washes of 15 min each in washing buffer (TBSplus 0.2 % Tween 20), the membranes were incubated inTBS containing goat anti-mouse IgG alkaline phosphate-conjugated secondary antibodies (1:10000 dilution). Themembranes were washed again (three times each for 15 min)with washing buffer and then treated with BCIP/NBTreagent (Sigma) for 10–30 min [18]. The protein bandsobtained were further subjected to densitometric analysis.The quantification of individual protein bands was done usingAlpha Ease FC 4.0.0 software (Alpha Innotech).

Statistical analysisThe results are presented as means +− S.D. for threeseparate experiments. Statistical ANOVA (three-wayANOVA) was performed for multiple comparisons followed

by a post hoc test using the statistical software SPSS,version 11. Significance was tested and P < 0.05 wasconsidered significant.

Results

Cytotoxic/proliferative effect of REC-2006 onHepG2 cellsTo evaluate the cytotoxic and cell-proliferative effects ofREC-2006 on HepG2 cell line, an MTT assay was carriedout as described in the Materials and methods section. REC-2006 treatment showed an LC50 of 10−1μg/ml. However,cell proliferative activity of REC-2006 started at a 100-foldlower concentration range (10−3–10−7μg/ml). The prolife-rative concentration range of REC-2006 was used for testingthe radioprotective efficacy in HepG2 cells.

Effect of γ -radiation and the radioprotective effectof REC-2006 on HepG2 cell lineTo evaluate the effect of γ -radiation and the radioprotectiveefficacy of REC-2006 on HepG2 cells, colony formation assaywas carried out. HepG2 cells were plated in a T-25 cultureflask and allowed to attach. The attached HepG2 cellswere exposed to different radiation doses and were furtherincubated to develop the colony as described in the Materialsand methods section. HepG2 cells exhibited the value ofLD50 at 7.5 Gy; however, 80 +− 2% mortality (LD80) wasobserved at 10 Gy (Figure 1B). The DMF (dose modificationfactor) for REC-2006 was calculated as 1.72 +− 0.2 and4 +− 0.3 at radiation LD50 and LD80 respectively (Figure 1A).REC-2006 treatment exhibited maximum protection of80 +− 3% at the concentration of 10−5μg/ml when treated2 h before acute irradiation (10 Gy) (LD80; 10 Gy).

Effect of PFT on REC-2006-mediatedradioprotection in HepG2 cellsTo evaluate the effect of p53 inhibition on REC-2006-mediated radioprotection, HepG2 cells were pretreatedwith PFT (10 mM) along with REC-2006 (10−5μg/ml)and further challenged with an acute radiation dose(10 Gy). The results of survival studies showed that thePFT (10 mM) treatment decreased the REC-2006-mediatedsurvival (radioprotection) of HepG2 cells by 33 +− 2 % whenchallenged with acute irradiation (10 Gy), as comparedwith survival percentage obtained when only REC-2006treatment was given 2 h before irradiation (Figure 1B,columns 3 and 5). PFT treatment alone increased theradiosensitivity of HepG2 cells (Figure 1B, columns 2and 4).

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Pifithrin-α-mediated decrease in radioprotection 57

Figure 1 Radiation toxicity and survival/protection in HepG2 cell lines

(A) Effect of γ-radiation and REC-2006 on HepG2 cells; (B) effect of PFT onREC-2006-mediated radioprotection.

DNA protection against lethal irradiation byREC-2006To evaluate the protection of DNA from lethal irradiation,single-cell gel electrophoresis (comet assay) was performedat 2, 4, 8 and 10 h after final treatment. Considerabledifferences in percentage DNA in comet tail wereobserved in radiation only and REC-2006 (−2 h + radiation)treatment groups in HepG2 cells at 4 h; however, thedifferences widened up to 8 h and, after that, nodiscernible changes in differences of percentage DNA wereobserved in comet tail of radiation only and REC-2006(−2 h + radiation) treatment groups (Figure 2).

Maximum DNA damage was observed in irradiated(10 Gy) HepG2 cells; however, radiation-mediated DNAdamage was found to be better repaired at 8 h in HepG2cells when pretreated with REC-2006 (Figure 2, column 3).Quantitative analysis of DNA indicated the presence ofalmost 50% DNA in the comet tail of radiation-treatedHepG2 cells; however, only 20 +− 3% DNA was found after8 h in the comet tail of REC-2006 (10−5 μg/ml) + radiation-treated HepG2 cells (Figure 2, columns 3 and 4).

Figure 2 Effect of REC-2006 and γ -radiation on percentage DNA contentin comet tail of HepG2 cells at different time intervals

Modulation of pro-apoptotic and anti-apoptoticprotein expression upon REC-2006, PFT andradiation treatmentTo evaluate the effects of REC-2006, PFT and γ -radiationon pro- and anti-apoptotic proteins, the expressions of p53,Mdm2, Hsp70, HSF1, Bcl-2 and Ras-GAP were evaluated byimmunobloting. A significant (P < 0.05%) increase (40 +− 3%)in p53 expression was observed in HepG2 cells treatedwith REC-2006 as compared with the untreated control(Figure 3A, lanes 1 and 2). Maximum induction (77 +− 4%increase) of p53 protein expression was observed in theirradiated HepG2 cells as compared with untreated control(Figure 3A, lanes 1 and 3), whereas radiation-induced p53expression was found to be significantly (P < 0.05%) reducedin the HepG2 cells treated with REC-2006 at 2 h beforeirradiation as compared with the irradiated HepG2 control(Figure 3A, lane 3 and 4). PFT treatment significantly(P < 0.05) inhibited the expression of p53 protein in alltreatment groups (Figure 3A, lanes 1–4). The expressionof p53 was observed only in the radiation-treated group ofPFT-pretreated HepG2 cells (Figure 3A, lane 3).

The expression of Mdm2 was found to be stronglyinhibited in radiation-treated group of HepG2 cells, ascompared with the untreated control (Figure 3B, lanes 1and 3). However, the expression of Mdm2 was observedto be significantly (P < 0.05) enhanced (by 62 +− 4%) inHepG2 cells treated with REC-2006 2 h before irradiation,as compared with the irradiated control (Figure 3B, lanes 3and 4). PFT induced no quantitative change in the expressionlevel of Mdm2. REC-2006 treatment with or without PFTexhibited the same expression pattern of Mdm2 (Figure 3B,lanes 1–4).

REC-2006 treatment slightly induced the expressionof HSF1 as compared with the untreated control ofHepG2 cells (Figure 4A, lanes 1 and 2). Significantly higherincrease (30 +− 3%) in the expression of HSF1 was observedwhen REC-2006 was treated at 2 h before irradiationas compared with the only radiation treatment group in

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58 P. K. Singh and others

Figure 3 Relative expression of (A) p53 and (B) Mdm2 in differenttreatment groups of HepG2 cells

*P < 0.05, REC-2006 group compared with the untreated con-trol. $P < 0.05, γ-radiation group compared with untreated control.#P < 0.05, REC-2006 + γ-radiation group compared with γ-radiation group.§P < 0.05, with the PFT group compared with the without-PFT group.

HepG2 cells (Figure 4A, lanes 3 and 4). PFT reduced thequantitative expression of HSF1 in all treatment groups ofHepG2 cells (Figure 4A, lanes 1–4). A significant (P < 0.05)increase (19 +− 3%) in expression of Hsp70 was observedin REC-2006 alone as compared with the untreatedcontrol (Figure 4B, lanes 1 and 2). However, maximuminduction (increase by 61 +− 3%) in expression of Hsp70 wasobserved on REC-2006 treatment at 2 h before irradiationas compared with the only radiation treatment groupin HepG2 cells. PFT alone decreased the expression ofHsp70 by 18 +− 2% as compared with the untreated control(Figure 4B, lane 1). The expression of Hsp70 decreased (by28 +− 3%) when REC-2006 was treated with PFT in irradiatedHepG2 cells as compared with the PFT untreated REC-2006 + radiation treatment group (Figure 4B, lane 4).

A significant induction in Bcl-2 expression wasobserved in the HepG2 cells on REC-2006 treatment ascompared with the untreated control (Figure 4A, lanes 1and 2). An increase in the expression of Bcl-2 was observedin the REC-2006 (−2 h + radiation) treatment group as

Figure 4 Relative expression of (A) HSF1 and (B) Hsp70 in differenttreatment groups of HepG2 cells

*P < 0.05, REC-2006 group compared with untreated control. #P < 0.05,REC-2006 + γ-radiation group compared with γ-radiation group. §P < 0.05,with the PFT group compared with the without-PFT group.

compared with irradiated control (Figure 4A, lanes 3 and 4).PFT alone increased the expression of Bcl-2 as comparedwith the untreated control (Figure 4A, lane 1); however,PFT decreased the REC-2006-mediated expression of Bcl-2(by 32 +− 3%) in the REC-2006 + radiation treatment group(Figure 5A, lane 4). A significant (P < 0.05) enhancementin the expression of Ras-GAP was observed on REC-2006 treatment as compared with the untreated control(Figure 5B, lanes 1 and 2). Similarly, expression of Ras-GAPwas found to be significantly (P < 0.05) enhanced upon REC-2006 treatment (given 2 h before irradiation) as comparedwith the irradiated control (Figure 5B, lanes 3 and 4). PFTinduced no quantitative changes in the expression patternof Ras-GAP. The expression of Ras-GAP was found to benearly the same with and without PFT in any treatmentgroups (Figure 5B, lanes 1–4).

Influence of REC-2006 and PFT treatment oncell-cycle-regulatory proteinsTo evaluate the possible effect of REC-2006 and PFTtreatment on cell-cycle-regulatory proteins, immunoblot

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Figure 5 Relative expression of (A) Bcl-2 and (B) Ras-GAP in differenttreatment groups of HepG2 cells

*P < 0.05, REC-2006 group compared with untreated control. #P < 0.05,REC-2006 + γ-radiation group compared with γ-radiation group. §P < 0.05with PFT group compared with the without-PFT group.

analysis of cyclin D1, CDK4, cyclin B1 and p21waf-1/cip-1 wascarried out. A significant reduction (40 +− 3%) in the cyclinD1 expression was observed in HepG2 cells treated withREC-2006 at 2 h before irradiation as compared with theonly irradiated HepG2 cells (Figure 6A, lanes 3 and 4),whereas, on the other hand, no such decrease was observedwhen REC-2006 was treated with PFT (lanes 3 and 4). WithPFT, 62 +− 3% more expression of cyclin D1 was observedin REC-2006 + radiation treatment group as compared withthe without PFT treatment group (Figure 6, lane 4). PFTtreatment increased the expression of cyclin D1 in theradiation treatment group as compared with the onlyirradiated control (Figure 6A, lane 3). Again no significantchange in the expression of cyclin D1 was observed inany of the treatment groups of HepG2 cells pretreatedwith PFT (Figure 6A, lanes 1–4). As a result of REC-2006treatment, expression of CDK4 was found to be decreased(14 +− 2%) in HepG2 cells as compared with untreatedcontrol (Figure 6B, lanes 1 and 2). Similarly, expressionof CDK4 was reduced (16 +− 3%) further on REC-2006treatment at 2 h before irradiation as compared with the

Figure 6 Relative expression of (A) cyclin D1 and (B) CDK4 in differenttreatment groups of HepG2 cells

*P < 0.05, REC-2006 group compared with untreated control. #P < 0.05,REC-2006 + γ-radiation group compared with γ-radiation group. §P < 0.05,with PFT group compared with the without-PFT group.

only irradiated HepG2 cells (Figure 6B, lanes 3 and 4). Nodecrease in the expression of CDK4 was observed whenPFT was treated with or without REC-2006 (Figure 6B,lanes 1 and 2). PFT treatment increased the expressionof CDK4 in radiation treatment group as compared withthe only irradiated control (Figure 6B, lane 3). In REC-2006 + radiation treatment group, the expression of CDK4was found to be much higher (42 +− 3%) with PFT treatmentas compared with without PFT treatment (Figure 6B,lane 4). Again, no significant change in the expression ofCDK4 was observed with any treatment groups of HepG2cells pretreated with PFT (Figure 6B, lanes 1–4).

A significant decrease (33 +− 3%) in the expression ofcyclin B1 was observed when REC-2006 was treated at2 h before irradiation as compared with the only irradiatedHepG2 control (Figure 7A, lanes 3 and 4). However, on theother hand, no decrease was observed in the correspondingtreatment groups when REC-2006 was treated with PFT(Figure 7A, lanes 3 and 4). REC-2006 alone increased(30 +− 2%) the expression of p21waf-1/cip-1 as compared withthe untreated control (Figure 7B, lanes 1 and 2). However, noexpression of p21 was observed in the PFT treated control

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60 P. K. Singh and others

Figure 7 Relative expression of (A) cyclin B1 and (B) p21waf-1/cip-1 in differenttreatment groups of HepG2 cells

*P < 0.05, REC-2006 group compared with the untreated control. #P < 0.05,REC-2006 + γ-radiation group compared with γ-radiation group. §P < 0.05,with PFT group compared with the without-PFT group.

and REC-2006 + PFT treatment groups (Figure 7B, lanes1 and 2). The expression of p21 was found to be enhanced(33 +− 3 %) in the REC-2006 + radiation treatment group ascompared with the only irradiated HepG2 cells (Figure 7B,lanes 3 and 4). PFT treatment significantly (P < 0.05) reducedthe expression of p21 in all the treatment groups (Figure 7B,lanes 1–4). Among the PFT treatment groups, p21 wasobserved only in the REC-2006 + radiation treatment group(Figure 7B, lane 4).

Effects of REC-2006 and PFT on the expression ofapoptosis effector/inducer proteinsTo evaluate the possible effect of PFT-mediated inhibitionof p53 on caspase 3 and Bax, HepG2 cells were treatedwith PFT prior to REC-2006 (with or without radiation)treatment. The results of the study showed no expressionof caspase 3 in the REC-2006-treated and only PFT-treatedcontrol groups (Figure 8A, lanes 1 and 2). The expression ofcaspase 3 decreased in the REC-2006 + radiation treatmentgroup as compared with only radiation-treated HepG2 cells(Figure 8A, lanes 3 and 4). PFT treatment slightly decreased

Figure 8 Relative expression of (A) caspase 3 and (B) Bax in differenttreatment groups of HepG2 cells

#P < 0.05, REC-2006 + γ-radiation group compared with the γ-radiationgroup. §P < 0.05, with PFT group compared with the without-PFT group.

the expression of caspase 3 in the REC-2006 + radiationtreatment group as compared with the PFT untreatedREC-2006 + radiation treatment group (Figure 8A, lane 4).No expression of Bax was observed in the REC-2006-treated and only PFT-treated control groups (Figure 8B,lanes 1 and 2). The expression of Bax decreased in the REC-2006 + radiation treatment group as compared with theonly irradiated HepG2 cells (Figure 8A, lanes 3 and 4). PFTtreatment significantly (P < 0.05) increased the expressionof Bax in the REC-2006 + radiation treatment group ascompared with the PFT untreated REC-2006 + radiationtreatment group (Figure 8A, lane 4).

Discussion

P. hexandrum Royle has been used since time immemorialin the Indian and Chinese systems of medicine for thetreatment of various diseases and disorders [23]. The radio-protective properties of P. hexandrum have been reportedpreviously by our Institute [12–19]. In the presentexperiments, the radioprotective effect of a chemicallycharacterized fraction of P. hexandrum (code REC-2006;

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Pifithrin-α-mediated decrease in radioprotection 61

containing podophyllotoxin and its derivatives such as 4′-D-methylpodophyllotoxin β-D-glucopyranoside) [14] and itsmodulation by PFT were studied. Radiation LD50 and LD80

were observed at 7.5 and 10 Gy respectively at a dose rateof 48.3 cGy/min (Figure 1B) in HepG2 cell line. An almostsimilar LD50 has been reported in HepG2 by Guan et al. [24].REC-2006 exhibited a higher radioprotective potential, asindicated by its DMF of 1.72 +− 0.2 and 4 +− 0.3 at radiationLD50 and LD80 respectively (Figure 1A). PFT alone inducedradiosensitivity in HepG2 cells (Figure 1B, lane 4), indicatingthe plausible role for p53 in radiation protection. Further,PFT, when treated with REC-2006, reduced the survival ofHepG2 cells (Figure 1B, lane 5), again suggesting the probableinvolvement of p53 protein in REC-2006-mediated radiationprotection. The result of the comet assay indicated that theDNA repair/protective efficacy of REC-2006 declines after8 h in HepG2 cell line (Figure 2). Therefore immunoblotingexperiments were carried out on the protein samples taken8 h after the final treatment.

The expression of p53 increased in the REC-2006treatment group as compared with the untreated control(Figure 3A, lanes 1 and 2); however, it decreased in the REC-2006 + radiation treatment group as compared with the onlyirradiated control (Figure 3A, lanes 3 and 4). Again, PFT-mediated inhibition of p53 reduced the REC-2006-mediatedradiation protection in HepG2 cells (Figure 1B, lanes 3 and5), which further suggested that not only the availability ofp53, but also its optimum level, is mandatory to achieve themaximum DNA repair/protection against lethal irradiation.Our results corroborate with those of Mallya and Sikpi [25]who reported the involvement of p53 in γ -radiation-inducedDNA repair in human lymphoblasts [25]. The expression ofMdm2, as observed in HepG2 cells, decreased in the REC-2006 treatment group (Figure 3B, lanes 1 and 2); however, itincreased on REC-2006 treatment at 2 h before irradiationas compared with the only irradiated HepG2 cells, suggestingthat REC-2006 helps in maintaining the threshold level ofp53 protein to deliver its anti-apoptotic and pro-survivalfunction by modulating the expression of Mdm2. The Mdm2protein has been shown to inhibit p53 activity by bindingto the p53 transactivation domain and interfering with therecruitment of basal transcription machinery components[26]. In addition, regulation of p53 response to DNA damageby Mdm2-mediated ubiquitination has also been reportedpreviously, supporting the results of our investigation[27]. No discernible changes in Mdm2 expression in therespective PFT treated and untreated groups suggestedthat PFT did not interfere with the p53-mediated feedbackinduction of Mdm2 expression in HepG2 cells. Theconcomitant increase in HSF1 and Hsp70 expression in theREC-2006 + radiation treatment group as compared withthe irradiated control indicated that REC-2006 increasedHsp70 expression by increasing the expression of HSF1 and

thereby increasing the survival of HepG2 cells. Increasedexpression of Hsp70 has been shown to protect the cellfrom apoptosis by inhibiting the release of cytochromec from mitochondria [28], by inhibiting Apaf-1 (apoptoticprotease-activating factor 1) [29], by antagonizing AIF(apoptosis-inducing factor) expression [17,30] and byinhibiting nuclear import of AIF [31]. PFT reduced theexpression of Hsp70 in the REC-2006 + radiation treatmentgroup (Figure 4A, lane 4) by decreasing the expressionof HSF1 and thereby reducing the REC-2006-mediatedsurvival in HepG2 cells. REC-2006 treatment at 2 h beforeirradiation up-regulated the expression of Bcl-2 and Ras-GAP as compared with the irradiated control (Figures 5Aand 5B, lanes 3 and 4); however, PFT treatment decreasedthe expression of Bcl-2 in the REC-2006 + radiationtreatment group (Figure 5A, lane 4) and induced no change inthe expression of Ras-GAP, indicating that PFT inhibits theREC-2006-induced pro-survival genes mainly by inhibitingthe expression of Hsp70. PFT-mediated inhibition of HSPs[32] and an Hsp70-mediated increase in Bcl-2 expressionfurther rendered support to our observations.

Regulation of the cell cycle at various phases afterirradiation decides whether cells will survive or not.Therefore, the role of PFT and REC-2006 in modulationof cell-cycle-regulatory proteins (i.e. cyclin D1, CDK4,cyclin B1 and p21) and their implications in radioprotectionwere studied. The pRb (retinoblastoma protein) proteinnegatively regulates the G1–S transition by binding to the E2Ftranscription factor, which is required for DNA replicationin S-phase [33]. Cyclin D1/CDK4 phosphorylates pRb andtherefore releases the E2F transcription factor prior to thecell traversing the G1 checkpoint and entering S-phase [34].Therefore a sharp decrease in the expression of cyclinD1 and CDK4, as observed on REC-2006 + irradiationtreatment, indicated that REC-2006 induced cell-cycle arrestin G1-phase of the cell cycle and thereby provided sufficienttime for the cells to repair damage. REC-2006-mediateddown-regulation of cyclin D1 as well as a leap in G1-phasecan also be explained by the observation of up-regulation ofHsp70 in REC-2006 + radiation treatment group. Hsp70-induced down-regulation of cyclin D1 and inhibition ofγ -radiation-induced cell death have been reportedpreviously by Lee et al. [35]. PFT decreases the expression ofHsp70 as well as HSF1 [32], and therefore PFT treatment isunable to induce G1-phase arrest and consequently lead toγ -radiation-induced cell death. Mild decrease in cyclin B1expression in the REC-2006 + radiation treatment group(Figure 7A, lanes 3 and 4) explained the REC-2006-inducedG2-phase arrest in HepG2 cells. However, on the otherhand, no such decrease in the expression of cyclin B1 wasobserved in the corresponding treatment groups when REC-2006 was treated with PFT (Figure 7A, lane 4) indicatingthe role of p53 in cyclin B1-induced transient G2-phase

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62 P. K. Singh and others

Figure 9 Schematic diagram showing REC-2006-mediated radioprotection and its inhibition by PFT

arrest leading to radiation protection. Lower accumulationof cyclin B1 and a lower frequency of radiation-inducedmitotic catastrophe in wild-type p53-expressing cells [36]further favoured the results of the present study. REC-2006-mediated G1 arrest and decrease in the expression of cyclinD1 and CDK4 can also be explained by increase in theexpression of p21waf-1/cip-1

, which is a downstream proteindirectly regulated by p53 protein. REC-2006-mediatedinduction of cell-cycle arrest in G1-phase can also beexplained by previous findings that the p21 protein regulatesG1 arrest by binding with PCNA (proliferating-cell nuclearantigen) that restricts its ability to activate DNA polymerase-δ (which is a vital protein for DNA synthesis in S-phase) [37].REC-2006-mediated increase in p53 expression and down-regulation of cell-cycle progression in G1/S-phase leadingto radiation protection can be corroborated by previousfindings with WR1065 (a well-known radioprotector), whichrenders radioprotection by activating p21waf-1 and down-regulating cell-cycle progression through a p53-dependentpathway [38]. As PFT inhibited the expression of p53 protein[9–11,32], and thereby the expression of p21, PFT treatmentmight therefore have allowed the radiation-damaged cellsto proceed from G1-phase to S-phase, resulting in theincorporation of damaged DNA into the next generation,leading to cell death.

To determine the possible effect of PFT on apoptosiseffector/inducer proteins, we evaluated the expression of

caspase 3 and Bax. No expression of caspase 3 and Baxwas observed in the REC-2006-treated and the PFT-treatedcontrol groups (Figure 8A, lanes 1 and 2), indicating thatPFT (10 mM) and REC-2006 (10−5μg/ml) are non-toxic toHepG2 cells. The expression of caspase 3 as well as Baxdecreased in the REC-2006 + radiation treatment group ascompared with irradiated HepG2 cells (Figures 8A and 8B,lane 3 and 4), indicating the anti-apoptotic function of REC-2006, which eventually resulted in radiation protection. PFTtreatment slightly decreased the expression of caspase 3and significantly (P < 0.05) increased the expression of Baxin the REC-2006 + radiation treatment group as comparedwith the PFT untreated REC-2006 + radiation treatmentgroup (Figure 8A, lane 4), indicating that PFT decreasesREC-2006-mediated radiation protection by inducing theexpression of Bax. Again, decrease in Bax expression andconsequent inhibition of apoptosis by REC-2006 treatmentcan be explained by concomitant increase in the expressionof Hsp70 in the REC-2006 + radiation treatment group.The PFT-mediated decrease in expression of Hsp70 in theREC-2006 + radiation treatment group (Figure 4B, lane 4)resulted in increased Bax expression (Figure 8B,lane 4) and led to apoptosis and consequently reduced theradioprotective efficacy of REC-2006 in HepG2 cells.

It can be concluded that p53 protein is an essen-tial component for achieving the REC-2006-mediatedprotection against acute irradiation in HepG2 cells. Higher

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Pifithrin-α-mediated decrease in radioprotection 63

radiation protection offered by REC-2006 in HepG2 cellsmight be an outcome of the cumulative action of inducedp53 expression, which contributed to cell-cycle arrest inthe G1- and G2-phases and rendered sufficient time forDNA repair. REC-2006 rendered radioprotective effect,inducing the Hsp70 expression, enhancing the pro-survivalproteins such as Bcl-2 and Ras-GAP and inhibiting theapoptosis inducer/effector proteins such as caspase 3 andBax. However, PFT, as depicted schematically in Figure 9,reduced the radioprotective efficacy of REC-2006 (i) by in-hibiting the expression of Hsp70 and thereby the expressionof Bcl-2, (ii) by up-regulating the cell-cycle-regulatoryproteins and thereby reducing the span of time for DNArepair and (iii) by inducing Bax-mediated apoptosis.

Further experiments are being carried out to elucidateREC-2006-mediated radiation protection in p53-negativeHep3B cell line as an experimental model.

Acknowledgement

We thank the Director of INMAS for providing us withnecessary facilities.

Funding

This work was supported by the Defence Researchand Development Organization, Ministry of Defence,Government of India; the Indian Council of Medical Research(Delhi, India) (Research Fellowship to P.K.S.)

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Received 17 November 2008/6 April 2009; accepted 1 May 2009Published as Immediate Publication 1 May 2009, doi:10.1042/BA20080250

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